Neuromedin U receptor NMUR2 and nucleotides encoding it

ABSTRACT

A new neuromedin U receptor, designated NMUR2 has been found, which is involved in modulation of feeding behavior in mammals. Ligands of this receptor are able to modulate eating, and weight gain. Amino acid sequences of the human and rat forms, as well as their nucleic acid sequences are given.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a § 371 National Stage Application of PCT/US01/13386, international filing date of Apr. 25, 2001, which claims priority to U.S. Ser. No. 60/200,718, filed Apr. 27, 2000, now expired.

FIELD OF THE INVENTION

This invention relates to new human and rat neuromedin U receptors, designated hNMUR2, and rNMUR2, to nucleic acids encoding them, and to use of them in various assays.

BACKGROUND OF THE INVENTION

Neuromedin U (NMU) is a neuropeptide that is widely distributed in the gut and central nervous system, particularly in brain regions implicated in the control of feeding behavior. NMU belongs to the broad class of neuropeptides first isolated from porcine spinal cord and later from other species with potent activity on smooth muscle. One orphan receptor designated FM-3 (now NMUR1) was previously identified as a high affinity receptor of NMU, which is the subject of U.S. Provisional Patent Application Ser. No. 60/092,623 (filed Jul. 13, 1998) and International Patent Application No. PCT/US99/15941 (filed Jul. 13, 1999). NMU, when injected into the rat brain, caused a marked suppression of food intake. Thus it appears that ligands of neuromedin receptors have potential as drugs which modulate feeding and regulate weight. However, it is equally clear that NMUR1 is not the only receptor whose activity is responsible for eating behaviors.

It would be desirable to further identify and characterize other receptors whose ligands are potential drugs for eating disorders.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention is a novel human receptor, designated hNMUR2 (SEQ.ID.NO. 2), free from associated proteins. This invention also relates to various functional domains of this receptor, such as the extracellular domain and the intracellular domain, and to hybrid molecules comprising at least one of these sequences. Also part of this invention are nucleic acids which encode this receptor, vectors such as viral vectors, plasmids and the like, which comprise these nucleic acid sequences, and host cells which comprise the vectors. In preferred embodiments, the nucleic acid is DNA, and especially cDNA.

Another aspect of this invention a method to identify compounds which modulate the feeding activity of a mammal comprising:

contacting the compound and a NMUR2 receptor; and

-   -   determining if activity of the NMUR2 receptor is modulated.

Another aspect of this invention is the rat homologue of the human receptor (designated rNMUR2), which is free from associated proteins (SEQ.ID.NO. 6.). This invention also relates to various functional domains of this receptor, such as the extracellular domain and the intracellular domain, and to hybrid molecules comprising at least one of these sequences. Another aspect of this invention is a nucleic acid which encodes the rNMUR2 receptor; in preferred embodiments the nucleic acid is DNA, and is preferably cDNA. Yet another aspect of this invention are vectors, such as plasmids, viral vectors, and the like which comprise a rNMUR2 gene. Still another aspect of this invention are host cells which comprise a vector carrying a rNMUR2 gene.

DESCRIPTION OF THE FIGURES

FIG. 1 is the cDNA sequence of human NMUR2 (SEQ.ID.NO. 1).

FIG. 2 is the predicted polypeptide sequence of human NMUR2 (SEQ. ID. NO. 2).

FIG. 3 is the translation of the open reading frame of human NMUR2 (SEQ.ID.NOS. 3 and 4).

FIG. 4 is the cDNA sequence of rat NMUR2 (SEQ.ID.NO. 5)

FIG. 5 is the predicted polypeptide sequence of rat NMUR2 (SEQ.ID.NO. 6).

FIG. 6 is the translation of the open reading frame of rat NMUR2 (SEQ.ID.NOS. 7 and 8).

FIG. 7 is the amino acid sequences and alignments of human, rat and porcine neuromedin U (SEQ.ID.NOS. 9, 10, 11, and 12)

FIG. 8 shows the alignment of human NMUR2 protein and rat NMR2 protein.

FIGS. 9A and 9B show functional activation of NMUR2 by NMU. FIG. 9A is NMUR2 in the aequorin assay using HEK293/aeq17 cells transiently transfected with human NMUR2. FIG. 9B is NMUR2 in the FLIPR assay using COS-7 cells transiently transfected with human NMUR2. In the FLIPR assay, total fluorescence was normalized to the maximum amount of fluorescence detected in the presence of the calcium ionophore A23187. (▾) porcine NMU-8; (▪) human NMU-25; (▴) rat NMU-23; (♦) porcine NMU-25. All the assays are shown as the means (+/−SEM) of triplicate determinations.

FIGS. 10A and 10B are in situ hybridization analysis of NMUR2 in the rat brain using ³³P-labeled anti-sense oligonucleotide probe specific for rat NMUR2, showing specific expression of NMUR2 in the PVN (paraventricular nucleus of the hypothalamus), Ep (ependymal layer in the wall of the third ventricle), and CA1 layer of the hippocampus. The signals were completely blocked in the presence of 100-fold molar excess of unlabeled probe.

FIGS. 11A and 11B are in situ hybridization analysis of NMU in the rat brain. FIG. 11A shows localization of NMU mRNA in coronal brain sections using ³³P-labeled anti-sense oligonucleotide probe specific for the gene encoding NMU. ARC: arcuate nucleus; ME: median eminence. The signals were completely blocked in the presence of 100-fold molar excess of unlabeled probe. FIG. 11B shows a decrease of NMU mRNA in the ventromedial hypothalamic area in rats fasted for 48 hours. Data shown are means (±SEM) of three experiments. *P<0.05, student t test.

FIGS. 12A–F show the effect of ICV-administrated NMU on food intake and other behaviors in rats. FIG. 12A shows the effect on overnight food intake. Food intake, expressed as percentage of control group, was significantly decreased in rats injected with 3 μg (−38±6%, n=12 per group) and 10 μg (−32±3%, n=12 per group) of NMU (ANOVA, F(3) 8.4, P=0.0002), and in rats injected with the positive control melanocortin agonist MT-II (0.3 μg; t(28)10.2, P<0.01). ** Scheffe post hoc analysis, P<0.01. FIG. 12B shows effect on cumulative feeding duration. Feeding duration was significantly decreased in rats injected with NMU either at 3 μg (−33%) or 10 μg (−39%) or with the positive control MT-II (−71%). FIG. 12C is core temperature change. A transient increase in core temperature was seen in the 3 μg NMU group that started about 40 min. post-dosing and lasted for approximately one hour. FIG. 12D is change in gross motor activity in rats in the fist hour post dosing. Activity was measured for 24 hours after NMU administration and compared to those of the same period of the pre-treatment. Gross motor activity was increased only in the first hour post-dosing and then returned to their pre-treatment levels in rats injected with either 1 or 3 μg of NMU. **, P<0.02. FIG. 12E is taste aversion. NMU at either 3 or 10 μg did not decrease saccharin intake relative to total intake at 24 hours post-dosing in a conditioned taste aversion assay. LiCl, an emetic control, decreased saccharin intake. [t test: t(6) 3.2, **, P=0.02]. FIG. 12F is sodium appetite. NMU at either 3 or 10 μg did not significantly change the total amount of salt intake while LiCl significantly decreased salt intake. [t test: t(4) 5.0, **, P=0.008].

FIG. 13 shows the various domains of human NMUR2 (SEQ.ID.NO. 2). The seven transmembrane domains (TM 1–7) are underlined. The sequence upstream of TM-1 is an extracellular domain, while sequences downstream of TM-7 is an intracellular domain.

FIG. 14 shows the various domains of rat NMUR2 (SEQ.ID.NO. 6). The seven transmembrane domains (TM 1–7) are underlined. The sequence upstream of TM-1 is an extracellular domain, while sequences downstream of TM-7 is an intracellular domain.

As used within the specification and claims the following definitions apply:

FM-3 (also designated NMUR1) is a previously identified human neuromedin U receptor, subject of U.S. Provisional Patent Application Ser. No. 60/092,623 (filed Jul. 13, 1998) and International Patent Application No. PCT/US99/15941 (filed Jul. 13, 1999).

NMUR2 (also designated FM-4) is a second neuromedin U receptor which plays a role in modulating the feeding behavior of a mammal. As used throughout “NMUR2” is not meant to refer to any particular origin of the NMUR2. “hNMUR2” means human NMUR2; “rNMUR2” means rat NMUR2.

NMU means neuromedin U.

“Free from associated protein” means that the receptor is not a naturally occurring NMUR2 receptor bound to its natural cell membrane.

A gene sequence and deduced amino acid sequence of a human orphan receptor was disclosed in WO 99/55732, published Nov. 4, 1999 (assigned to Astra Pharma, Inc.), and hereby incorporated by reference. Based on its structural similarity to the neurotensin receptor, this orphan receptor was designated NLR (neurotensin-like receptor), and it was hypothesized that its ligands would be useful agents for producing anesthesia and analgesia. The receptors of this invention share some gross structural similarity to the NLR receptor—both are the same length, but the human NMUR2 has six amino acids which differ from the NLR receptor:

Amino Acid Position NLR NMUR2 271 Leucine Phenylalanine 298 Threonine Serine 315 Leucine Phenylalanine 371 Serine Phenylalanine 383 Leucine Proline 388 Valine Methionine These six amino acid differences may contribute to NMR2's different activity. NMUR2 is involved with modulation of feeding behavior rather than anesthesia and analgesia.

Thus, one aspect of this invention is a method for identifying a compound which modulates feeding activity or weight of a mammal comprising:

a) contacting a cell comprising NMUR2 with the compound;

b) determining if the compound modulates NMUR2 activity.

Preferably the NMUR2 is recombinantly expressed in the cell. It may be introduced into the cell by conventional genetic engineering techniques, such as by conventional vectors including plasmids. Alternatively a cell line may be created which expresses NMUR2 in a non-transient fashion. Any host cell which is convenient may be used in these assays, preferably a human cell when the NMUR2 is the human NMUR2. Examples of suitable cell lines include 293 cells.

NMUR2 activity modulation can be determined in a number of ways. It may be a qualitative determination, i.e. a “positive” verses “negative” response. Alternately, the modulation can be quantified. Control systems may also be used, such as cells which are either mock-transfected and exposed to the putative ligand, or NMUR2 transfected cells which are exposed to a known negative or positive ligand.

In general, modulation of a receptor activity may be determined using a transactivation assay. In this assay, a “reporter construct” is introduced into a cell, which expresses either a recombinant receptor, or an endogenous receptor. The reporter construct comprises a reporter gene encoding a protein whose transcription and/or translation is easily measured, including such genes as β-galactosidase, luciferase, aequolorin, CAT, and the like. Upstream is a promoter (either the promoter naturally associated with the reporter gene, or a heterologous promoter) and upstream of the promoter is an activation sequence. When a ligand binds to the receptor, a cascade of intracellular reactions occur, and the result is that a binding protein binds to the activation sequence, activating the promoter, and transcription and translation of the reporter gene occurs. Such assays are described in U.S. Pat. No. 5,401,629, which is hereby incorporated by reference.

The cell line used in this assay is preferably a mammalian cell line, more preferably a human cell line. In one preferred embodiment the cell line is HEK293/aeq17, a human embryonic kidney cell line which contains an aqueorlin reporter gene. It is described in Button et al 1993 Cell Calcium 14:663–671, which is hereby incorporated by reference.

Another assay which is part of this invention is a FLIPR (Fluorometric Imaging Plate Reader) assay which monitors changes of intracellular Ca²⁺ concentration in real time. Thus another aspect of this invention is a method of identifying compounds which modulate the feeding behavior of an individual comprising: contacting cells expressing NMUR2 receptors with a compound; and determining changes in intracellular Ca⁺² concentration. In these assays, human, porcine and rat NMU activated NMUR2 with high affinity, and lead to Ca⁺² mobilization.

Another assay contemplated by this invention is a method of identifying compounds which modulate feeding behavior in an individual by a) contacting the compound and a NMUR2, and determining if binding occurs. In these assays, whole cells expressing the NMUR2 receptor are not necessary. While they can be used, membrane preparations, lysed cells or any other preparation containing receptors will suffice. Binding may be determined by monitoring behavior of a labeled ligand, such as ¹²⁵I-NMU-23 or appropriately labeled compound.

Rat NMUR2

Another aspect of this invention is the rat homologue of human NMUR2, and nucleic acids encoding this sequence. Rat NMUR2 was isolated using degenerate PCR on rat genomic DNA followed by genomic walking and PCR from rat cDNA. The rat gene was identified in genomic DNA. The open reading frame of rat NMUR2 encodes a protein of 395 amino acids, and is approximately 80% identical to the human NMUR2. The rNMUR2 can be used in assays in the same was as hNMUR2.

Another aspect of this invention are active fragments of NMUR2. These proteins are G-coupled proteins, exhibiting the classic 7-transmembrane domain structure (see FIGS. 13 and 14). Thus this invention includes active fragments, such as the extracellular domain which contains the binding region, which may, alone be used in binding assays for ligands, or which may be coupled to at least one domain from another receptor, creating a hybrid receptor. Additionally hybrid receptors can be created which utilize the intracellular domain on NMUR2 and at least one other region from a different receptor. Hybrids between the rat/human sequences are also included as part of this invention.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLES Example 1 Cloning of Human NMUR2

Genbank sequences were searched for sequences potentially encoding novel G protein-coupled receptors using the FAST_PAN data display tool (Retief, J.et al 1999 Genome Res 9:373–382, which is hereby incorporated by reference).

The genomic sequence AC008571 (Genbank accession number) contained a putative gene, preliminarily termed FM-4 that is approximately 51% identical to NMUR1 (both of which are hereby incorporated by reference).

Two primers, FM-4.F1 (5′-GAA ACA GAG CCT CGT ACC A-3′) (SEQ.ID.NO. 13) and FM-4.R1 (AGT CGG ATC CAA TTC AGG TTT TGT TAA AGT GGA) (SEQ.ID.NO. 14) were synthesized and used to amplify the full-length coding sequence of FM-4 from human testis cDNA. The PCR product was cloned into the vector pCRII (Invitrogen, Inc.), sequenced, and subcloned into the mammalian expression vector pcDNA3.1(−) (Invitrogen, Inc.). It was subsequently renamed NMUR2.

Example 2 Isolation of Rat Orthologs of NMUR2

For the isolation of rat NMUR2, two degenerate primers (forward): 5′-TTC AGC CTG GCN GTN TCN GA-3′ (SEQ.ID.NO. 15) and (reverse): 5′-GCT GAG GAT NGA NGC RAA RCA-3′ (SEQ.ID.NO. 16) were used to carry out PCR reactions on rat genoric DNA. The resulting PCR product was subdloned into pCRII and four independent clones were sequenced. Specific primers were synthesized and used to carry out genomic walking. Sequences corresponding to the start and stop codons of human NMUR2 were identified, and PCR primers flanking the coding sequence were used to amplify the full-length open reading from rat stomach cDNA. The PCR product was cloned into pCRII and sequenced.

Example 3 Generation of NMUR2-Expressing Cells

The complete coding sequence of hNMUR2 was subcloned into the expression vector pIRESpuromycin (Clontech, Inc., Palo Alto, Calif., USA). The plasmid hFM4/pIRESpuro was then transfected into HEK293/aeq17 cells (Button and Brownstein, 1993, Cell Calcium, 14:663–671) using Lipofectamine-2000 (Gaithersburg, Md., USA) and cells stable expressing hFM-4 were selected as described in Liu et al, 1999 Biochem. Biophys. Res. Commun. 266:174–178, which is hereby incorporated by reference.

Example 4 Aequorin Functional Assays

The HEK293/aeq17 cell line was licensed from NIH (Button and Brownstein, 1993, Cell Calcium, 14:663–671). The cells were grown in Dulbecco's Modified Medium (DMEM, GIBCO-BRL, Gaithersburg, Md., USA)+10% fetal bovine serum (heat inactivated), 1 mM sodium pyruvate, 500 μg/ml Geneticin, 100 μg/ml streptomycin, and 100 units/ml penicillin. NMUR2/pIRESpuro plasmid DNA was transiently transfected into HEK293/aeq17 using Lipofectamine-2000 (Gaithersburg, Md., USA) following the conditions suggested by GIBCO-BRL. Twenty four hours after transfection, cells were washed once with DMEM+0.1% fetal bovine serum, and then charged for one hour at 37° C./5% CO₂ in DMEM containing 8 μM coelenterazine cp (Molecular Probes, Eugene, Oreg., USA) and 30 μM glutathione. The cells were then washed once with Versene (GIBCO-BRL, Gaithersburg, Md., USA), detached using Enzyme-free cell dissociation buffer (GIBCO-BRL, Gaithersburg, Md., USA), diluted into ECB (Ham's F12 nutrient mixture (GIBCO-BRL)+0.3 mM CaCl₂, 25 mM HEPES, pH7.3, 0.1% fetal bovine serum). The cell suspension was centrifuged at 500×g for 5 min. The supernatant was removed, and the pellet was then resuspended in 10 mL ECB. The cell density was determined by counting with a hemacytometer and adjusted to 500,000 cells/ml in ECB.

Human NMU-25 was custom synthesized by Research Genetics (Huntsville, Ala., USA). Rat NMU-23, porcine NMU-8, and porcine NMU-25 were purchased from Phoenix Pharmaceuticals (Belmont, Calif., USA). Results are shown in FIG. 9A.

Example 5 FLIPR Functional Assay

Cos-7 cells, grown in Dulbecco's Modified Medium (DMEM, GIBCO-BRL, Gaithersburg, Md., USA)+10% fetal bovine serum, were transfected with h NMUR2/pcDNA3.1 using Lipofectamine-2000 (GIBCO-BRL, Gaithersburg, Md., USA). Two days post transfection, the cells were detached and seeded into 96-well plates at approximately 10,000 cells/well. The next day, cells were loaded with Fluo-3 in the presence of 2.5 mM probenicid. After washing, the cells were treated with varying concentrations of NMU. Fluorescence output was measured by a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Inc.). Results are shown in FIG. 9B.

Example 6 Expression Analysis

Quantitative in situ hybridization analysis in the rat brain was carried out described previously (Guan, X. M.,et al,.1998. Brain Res Mol Brain Res 59, 273–279, which is hereby incorporated by reference). For rNMUR2, the probe used was ³³P-labeled anti-sense oligonucleotides (equal mix of oligo 420: 5′-AGG AAA GGG TAA TTG TGC CAC ATC TCG TAG ATT TCC AGA GGC ATC-3′(SEQ.ID. NO.17) and oligo 421: 5′-CAC AGT CTC GAA GAG GGC TGT CTT GAA GTA GCA TCC CAC AGG C-3′(SEQ.ID.NO.18)). For NMU, the probe used was ³³P-labeled anti-sense oligonucleotide: 5′-TTC TGG TGG TAA TCT TTG AGG CGA TAT TGG CGT ACC TCT GCA AGC-3′(SEQ.ID.NO.19). Results are shown in FIGS. 10A, 10B, 11A and 11B.

Example 7 Animal Studies

Male rats (Charles River Sprague Dawley) weighing 250–350 g were maintained in a temperature and humidity controlled facility with a 12 hour light/dark cycle (4:00 AM lights on). Rats were individually housed in custom designed shoebox cages on wire floors and fed ad libitum with fresh diet provided daily. The shoebox cage had an external, restricted access feeder assembly that allows the animal to place only its head through an opening in the feeder assembly to access a detachable clear plastic food drawer. Attached to the food drawers was an infrared feeding monitor that projects a beam across the drawer above the food (MiniMitter, Inc., Sun River, Oreg.). When the animal broke the infrared beam it caused a switch closure. An oscillator then sent off pulses (one pulse/second) and the total number of pulses indicated the length of time that the beam was broken which corresponds to the length of time spent feeding (recorded as feeding duration).

Cannulation and ICV administration were performed essentially as described in Murphy et al 1998 Neuropeptides 32:491–497, which is hereby incorporated by reference. After cannulation, rats were allowed to recover a minimum of seven days before injection with test compounds. All test substances were dissolved in artificial cerebral spinal fluid (aCSF). Rats were injected ICV with 1, 3, or 10 μg of rat NMU-23 (Phoenix Pharmaceuticals). Additional rats were injected ICV with either 0.3 or 0.03 μg of MT-II (Peninsula Laboratories) as a positive control for food intake suppression (melanocortin receptor agonist). One group of rats also had a radio transmitter placed in the peritoneal cavity for measurement of core body temperature and gross motor activity (MiniMitter, Inc., Sun River, Oreg.). Another group of ICV-cannulated rats were used in conditioned taste aversion (CTA) and sodium appetite (SA) aversion assays.

In the CTA study, rats were conditioned to two hour daily access to water, with access to water from two bottles for two hours each day for three days. On the fourth day, rats were given 0.15% saccharin for the two hour period instead of water and saccharin consumption measured. Rats were injected NMU-23 (0, 3, or 10 μg, ICV). LiCl was used as a positive control (0.15 M; 2 ml/kg, i.p.). On the fifth day, rats were given saccharin alone for the first hour, then water was added for the remaining 23 hours. Fluid consumption was measured at 1, 2, and 24 hours post injection. Aversion was assessed as a function of drinking preferences.

In the salt appetite assay, rats were given 0.5 M NaCl salt water to drink for three days along with food and regular water. After three days, two injections of furosemide (5 mg /0.2 ml, s.c.) were given at one hour apart to sodium-deplete the rats. Rats were then returned to salt-free water and given a sodium-deficient diet. Rats actively seek to defend their internal sodium levels. Consequently, when sodium is depleted, they will avidly drink salt solutions unless ill or nauseous. Twenty-four hours following furosemide administration, rats were given NMU (0, 3, or 10 μg, ICV), or LiCl (0.15 M, 2 ml/kg, i.p.) and given water and 0.5 M NaCl to drink. Fluid consumption was measured 1, 2, and 24 hours post dosing.

Results are shown in FIGS. 12A–F. All rodent studies described were conducted in accord with rules and guidelines of the Merck Research Laboratories Institutional Animal Care and Use Committee and the “Guidelines for the Care and Use of Laboratory Animals” [DHHS Publication No. (NIH) 85–23, revised 1985]. 

1. A method to identify compounds which modulate neuromedin U receptor 2 (NMUR2) activity comprising: (a) contacting the compound and a NMUR2 receptor as set forth in SEQ ID NO:2; and (b) determining if the activity of the NMUR2 receptor is modulated by measuring changes of intracellular calcium concentration. 